Means for analyzing microscopic particles and the like



1961 M. L. POLANYI ET AL 2,969,708

MEANS FOR ANALYZING MICROSCOPIC PARTICLES AND THE LIKE Filed April 3, 1957 4 Sheets-Sheet l INVENTORS M/CHAEL L. POLANV/ P M/A/ JAMES E JOHNSTON MORDEIV 6'. 5 BY L I ATTORNEYS 1961 M. L. POLANYI ET AL 2,969,703

MEANS FOR ANALYZING MICROSCOPIC PARTICLES AND THE LIKE m 'm ]N VENTORS MICHAEL L. POLANVI \IAAFES E \laHA/sro v 6 MCRDEN 6. BROWN /1tg BY Jan. 31, 1961 M. POLANYI ET AL 2,959,703

MEANS FOR ANALYZING MICROSCOPIC PARTICLES AND THE LIKE Filed April 3, 1957 4 Sheets-Sheet 5 3.0 a. 3.8 4.2 4.5 5.0 5.4 s.s-

D/AMETR- IN /4 Jan. 31, 1961 M. L. POLANYI ET AL 2,969,703

MEANS FOR ANALYZING MICROSCOPIC PARTICLES AND THE LIKE Filed April 5, 1957 4 Sheets-Sheet 4 DIAMETER IN/J- l8 cor e l DARK R/NG [N VENT 0R5 g" 1 0 MICHAEL L. POLANY/ JAMS E. JOHNS raAl United States Patent i MEANS FOR ANALYZING MICROSCOPIC PARTICLES AND THE LIKE Michael L. Polanyi, Webster, and James. E. Johnston, Southbridge, Mass, and Mortlenv G. Brown, Wood: stock, Conn, assignors to American Optical Company, Southbridge, Mass, a voluntary association of Massachusetts Filed Apr. 3, 1957-, Ser. No. 650,518

11 Claims. (CI. 38-14) This invention relates to an apparatus for. analyzing certain physical properties of a very large number. or

collection of very small or microscopic objects, particles or bodies either uniformly arranged; or distributed. at random but in fairly close relation to one another and with said bodies or the. like either separated by air or other transparent medium therefractive indexof which may be unknown. More particularly and in keeping with the above conditions, the. invention relates to an apparatus by which a pattern of scattered light, whether same be due todifiraction, dispersion, refraction, internal reflection or the like, or any combination thereof, may be produced and rapidly analyzed to accurately indicate values. of said physical properties.

Anex-ample. wherein the apparatus of the present invention finds real utility is in. the analyzation of c'ertain physicalproper-ties of human blood such as determining mean or average volume of individual red blood cells in a solution, and the refractive index thereof. I

It; will be appreciated, however, from the description which follows that while the apparatus and method described at some length herein are in the main directed to blood analysis, the invention, withminor changes and/or. adjustments as set forth below, may also be usedin, analyzing other collections of microscopic bodies, particles and the like.

The determination of the average. size or volume of Neverthless, it is 'very useful information for clinical and' diagnostic purposes when it can be obtained in an easy and accurate manner. Such information, however, heretofo're has not been easy to obtain. Not only do red corpuscles of differet blood samples vary greatly in num her and size, but also vary greatly in shape, in hemoglobin concentration and in cell size distribution, etc.; with the result that the problems of determining the values mentioned above so as to. have real meaning and accuracy fordifferent samples by earlier known methods and apparatus have been difiic'ult and, time consuming; and these values at best were often subject to such undesirable con ditions as errors in counting, errors in pipett'ing, errors in chamber. size, errors in dilution and the like. A V

The apparatus of the present invention on the other hand provides. a convenient, rapid and dependable manner by which. average red blood cell volume'and refractive index may be obtained; and from which such other useful information as cell diameter, and an indication of distribution of cell sizes in the specimen under consider ation may be readily obtained f i It is, accordingly, an object of. the. present invention to provide an apparatus by which a measure of average cell size, volume and refractive index may be, obtained; and from this information, used in combination with a hematocrit reading, an accurate estimate of the total number red cells per unit volume of whole blood may be more easily obtained than hasbeenpossible heretofore, byknown methods and apparatus. i M It is another object or the present invention to provide ice in such. an apparatus simultaneously operable means whereby permanent records or graphs of values of the scattered light at a series of displacement distancesindicative of average blood cell volume and refractive index may be obtained.

It is, also an object of the invention to provide anapparatus. by. which such. graphs. or. permanent records may be made and used together with. hematocrit values of blood samples to provide accurateindications as to the average number of red blood cells contained in unitvolumes of-the whole blood being analyzed.

It is an additional object of the invention to provide compact, inexpensive and accurate apparatus of the above character which may be arranged in portable form where,- by the previously mentioned information relative to red corpuscles or the like in solution or other collection of microscopic bodies scattering light may be easily and rapidly obtained at any convenient location.

Other objects and advantages. of the present invention will become apparent from the detailed description which follows when taken in conjunction with the accompanying drawings in which:

Fig. 1 is a schematic view of a preferred form of optical system and associated apparatus for carrying out the method of the present invention;

Fig. 2 is a sketch showing in, an enlarged and somewhat exaggerated manner geometric relationships of certain operative parts of the apparatus of Fig. 1 thereof;

Fig. 3 is'a sketch similar to Fig. 2 for use in obtaining a better understanding of the invention;

Fig. 4 is. a graph obtained by use. of the apparatus of Fig. .1;

Fig. 5 is a fragmentary view of a part of the apparatus of Big. 1 showing an annular light. aperture used therein;

Fig. 6 is a chart showing a theoretical curve of seattered light intensities plottedagainst a dimensionless parameter for use in interpretation of an actual curve like that of Fig. 4;

Fig. 7 is a chart showing values of p the retardation value of the light passing centrally through a spherical particle or the like plotted against Z and, Z values and also against 'min max ratio values;

Figs. 8 and 9 are twochart's for joint use in determining diameter of particle and An, the difie'r'ence in the retrace tive index of the particle and the refractive index of the associated dilution or suspension fluid; and

Fig. 10 is a graph from whichvalues of particle or cell volume and An index values may be directly obtained.

Referring to the drawings in detail aiidparticularly to Fig. 1, it will be seen that a preferred embodiment of the apparatus is indicated generally by the numeral 10 and comprises a transparent specimen holder 12 upon which may be positioned a small quantity of solution, suspension or the like containing a verylarge number of very small particles, such as red corpuscles in solution, to be analyzed. It is not only desirable for best results that this solution be of such dilution that no appreciable overlapping of particles occurs when light is being transmitted through a thin layer on'the'h'older 12' and in a direction substantially normal to the plane or the holder but also desirable that the particles be spaced sufficiently so that none of the light passing one cell will be disturbed byan adjacent cell.

The sample holder 12 is carried upon an apertured stage 14 which is arranged to move, as indicated by the double-headed arrow 16, in a direction parallel to the opical axis 18 of a compound optical system 20 between a near position 22 and a far position 24 during opera of e pp r tus, The optical system 20, as

3 shown, comprises a pair of condensing lenses 26 aligned with a light source 28, a first surface reflector 30 for folding the system and directing the convergent beam of light received from the condenser fully and evenly toward a pinhole aperture 32 in an opaque diaphragm 34. Preferably, the light source 28 is a mercury vapor lamp and in order that monochromatic light of a preselected wave length, in accordance with the kind of specimen being considered, may be supplied at the pinhole 32, a color filter 36 of proper absorptive characteristics is employed where convenient between said source and the pinhole 32. A pinhole having a diameter of 0.3 mm. is acceptable.

The illuminated pinhole 32, it will be noted, is so disposed in axial alignment with a comparatively long focus objective 38 of said optical system that light received from said pinhole will be formed into a convergent beam 39 which is imaged as a very small spot at a focal plane 44}. At the focal plane 40,21 plate 41 is located. This plate 41 carries an opaque coating 42 which is of such configuration as to provide an inner opaque circular disklike portion 44 and an outer encircling opaque portion 46 arranged in such concentric spaced relation to each other as to form a narrow annular slit or light aperture 48 having a predetermined mean radius R, and a slit width which for reasons to be later described is substantially equal to the width of the image of the pinhole 32 at plane 40. This annular aperture 48 is arranged in concentric relation with respect to the optical axis 18.

Thus it will be appreciated that substantially all light which passes through the illuminated pinhole 32 and which is transmitted by the objective 38 will impinge upon and be intercepted the opaque disk-like portion 44 unless deviated or otherwise affected by other means in its path. This undeviated light in Fig. 1 when no blood sample or specimen is in place upon the sample holderlZ is indicated by solid lines or rays 39a and 39b.

Dotted lines 51 on the other hand indicate rays which have been so deviated by an axial object point at the surface of the holder 12 as to pass through the annular aperture 48.

The sample holder 12 during normal use of the apparatus is intended, as stated above, to support a thin layer or smear of a specimen, for example, blood containing red corpulscles, in the light beam 39 so as to be analyzed. Of course this layer will be of a suflicient area as to fully intercept .thbeam 39 for any and all positions of adjustment of the sample holder 12. Accordingly, when the beam of light 39 is illuminating a specimen on the sample holder 12, part of this light'will be diffracted or scattered in varying quantities and different directions by'each of the many small particles contained in the illuminated area. Accordingly, a certain 'part of this scattered light from each illuminated small object will travel towards the annular aperture 48 for each and every position of adjustment of the holder 12 between near and far positions 22 and 24 respectively. A much larger portion of the light beam, on the other hand, is undeviated and will be intercepted or blocked by the opaque central portion 44.

It will be appreciated from the description of Fig. 2 which follows that for each and every position of the holder 12 from its near position to its far position, the illuminated area of :the layer may be considered merely as a collection of separate particles in 'which'each particle will in effect contribute a hollow cone of scattered light in' such directions as to' pass through the annular aperture 48.

. At any single position of adjustment of the layer, it should be noted, all light rays of such a cone will have substantiallyequal angles of deviation; and furthermore all cones from such an illuminated area will be for all practical purposes geometrically substantially equal to one another.

Accordingly, in axial alignment with the objective 38 and rearwardly of the annular aperture 48 and diaphragm plate 41 is positioned a pair of condensing lenses 50 for directing this transmitted light onto a diffusing plate 54 rearwardly thereof. The condensing lenses 50 are of such predetermined optical design and so located with refererence to the diffusing plate, the annular aperture and the sample holder 12 that for all positions of the holder 12 not only will all the light passing through the aper ture 48 be directed as a spot by lenses 50 toward the diffusing plate 54 but also so located that these lenses will have their best focus when the holder 12 is located intermediate the near and far positions 22 and 24. Thus, the out-of-focus spots produced by the scattered light upon the diffusing plate 54 for the near and far positions of the sample will be of substantially equal sizes.

Rearwardly of the diffusing plate 54 and suitably positioned with reference to the light received thereby is a light sensitive device, such as a photomultiplier 56, for receiving this diffused light and producing an electrical current which is proportional to the intensity of the light energy being received. The use of this current will be described more fully hereinafter.

While in the disclosure of Fig. 1, an opaque disk 44 has been shown, it should be understood that other means for preventing direct light from reaching the photocell could be provided. In fact, the opaque disk 44 could be of dull black and substantially completely absorbing, or could be specularly reflecting and tilted so as to direct the light intercepted thereby laterally outwardly of the system, or even could be made of opaque material and of some controlled configuration so as to admit and trap the direct light rays within recessed areas thereof, the forwardly extending wall portions of such a light trap serving to prevent scattering of the direct light so re ceived and thus avoiding detrimental conditions result ing therefrom. In such a light trap, of course, care should be taken to avoid reflection of any direct light directly back toward the specimen.

The sample holder 12 with a blood specimen thereon is intended, during use of the device, to occupy not only the near and far positions mentioned above but also all of the various positions therebetween. In order that the holder 12 will successively occupy these positions during consideration of a specimen, the support 14 in the present embodiment, is carried by a movable frame bar 58 which has formed on one side thereof a rack 60 adapted to mesh with a worm gear 62. The gear 62, in turn, is mechanically driven in known manner by electric motor 64 which is preferably arranged to move the sample holder 12 along the optical axis 18 at a slow uniform rate and in either direction from one extreme operative position to the other.

- A recording table 66 for supporting a sheet of graph paper 68, or the like, in operative relation with respect to a scribing element 70 of a recorder 72, may be coupled (as shown) directly to the movable frame 58 so as to have simultaneous movement therewith. Alternatively, some other ratio than a 1 to 1 ratio of movement of the holder 12 relative to the table 66 could be arranged, if desired, by suitable mechanical or electrical means of known construction. However, for simplicity of disclosure, a direct coupled arrangement is shown.

The scribing element 70 is carried by an actuating arm 73 extending outwardly from a sensitive electric actuator '74 of the recorder 72 in such a manner as to move back or forth, as indicated by the double-headed arrow 76, in accordance with the amount of current being received by actuator 74. The photomultiplier is connected to a source of electrical energy 78 in such a manner that when light energy is received thereby during operation of the apparatus, a current will flow through the photomultiplier and through an electrical circuit 80 in which a load resistor 82 is arranged. The current which flows through thephotomultiplier will be proportional to the intensity of the light energy being received thereby and thus the vo ta a opacros h ad, es st m y be. e me power amplifier 84 inknown mannenand the output current from this. amplifier, supplied ftotherecorder 72. (A .001 ampere full, scale. recording millimeter has been found to give satisfactory results.) Therecorder arrangement as shown in Fig. 1 is such as to cause the arm 73 to move inwardly from a zero position varying amounts in accordance with the amount of current flowing in circuit 80. In, order to correlate the current intensities withthe positionjofthe sample in the convergent, beam as it is movedjtoward or away, fror'nythe aperture 48, it is very desirable toJha' ve a reference mark or marks placed onthe graph paper and this may beaccomplished, for example, by having the sample holder actuate an electrical switch, or the like, to momentarily interrupt the output current from the amplifier 84 as a fixed point is reached during the travel of the sample holder.

The arrangement of parts in Fig. 1* is such that when no sampleison the specimen holder 12, no scattering of light will occur and thus no lightwill begsupplied to the photo tube 56. Consequently, nocurrent will follow in circuit 80. At such time, the arm 73 will remainin its outer or extended position indicating a zero amplitude. However, if a sample is placed upon the holder 12 and the holder is moved axially by the motor 64 from its far position 24 to its near position 22, the light which is being scattered by the specimen at various angles of deviation relative to the optical axis 18 and in varying amounts at these different angles will be caused to successively pass through the annular aperture 48; In fact, it should be noted that all of the light being transmitted through the aperture 48 at any single instant is in effect in the form of a hollow cone of light all light rays of which are deviated by substantially the same angular amount.

The relationship of the deviated and undeviated light rays provided by the structure of Fig. l is of importance. Accordingly, an enlarged and somewhat exaggerated Sketch is given in Fig. 2 in order that a clearer understanding of the invention may be obtained. In this sketch, it will be seen that undeviated light rays 39a and 39b define substantially the lateral limits of the beam 39 when no microscopic objects are atfecting the beam and these rays are directed so that the beam forms a small spot of light upon the opaque disc-like portion 44. Of course, at the same time the beam will function to illuminate a small area of a smear or thin layer ofmaterial such as indicated at 88 upon the sample holder 12; and this illumination of the layer will take place no matter in what operative position along the optical axisv 18 the holder 12 may be located.

It is well known that such a smear or specimen containing small objects will diffract or scatter light impinging thereon. Therefore, if an axially disposed particle 90 in the layer of solution 88 is considered, it will be appreciated that this particle located between near and far positions 22 and 24 and at a variable distance it from the focal plane 40 will variously scatter light and some of this light will be directed toward and through the annular aperture 48 at any distance h, as indicated by devi ated rays 90a and 90b. In fact, all of the light from axial particle 90 which is transmitted through the annular aperture 48 will have substantially the same angle of deviation with respect to the optical axis 18. If, on the other hand, an off-axis small object in the specimen 88 is considered, such as object 92 which in Fig. 2 is being illuminated by the converging light ray 39a, it will be appreciated that scattered light will variously radiate therefrom and this scattered light will include light rays 92a and 92b which travel toward diametrically opposite parts of the annular aperture 48. Even though small object specti-vely which are relativeto theundeviatedlight ray,

39g substantially equal. This is largely due to the fact that. undeviated lightuayv 39a is converging toward the optical axis 18 and thus. nearly equally bisects the in.- cluded angle betWeenraysQSZa and92b. It should also be notedth'at theseangular values, 6 and. 0 will be for all practical purposes very nearly equal to the angle 0.

Such a favorable conditionwould not prevail, however, if for. example parallel light were employed to illuminate the layer; for as. isclear from Fig. 3, the illuminating beam couldnotbe as large in diameter as'the annular aperture 4.8. and also the off-axis rays of the illuminating beam would, not provide substantially equal amounts of deviation, for all scattered light from any small, illuminated object. to the annular aperture 48, This will bea'ppreciated if elfaxis small object 93, for instance, illuminated by. one of theparallel rays 94, is considered. Clearly the angles 0 and 0 indicating the amounts of deviation of scattered light rays 93a and 93b respectively which pass through annular aperture 48 are not equalto each other. Nor is either substantially equal to 0. Accordingly, such a mixture of deviations from all small objects in the illuminated area at any operative location of the axially movable support 12 would not give informationwhich can be readily used to advantage in analyzing the specimen being considered.

A distinct advantage is gained by use of the optical arrangement of Fig. 1 over any arrangement whereby the diffracted or scattered light is measured by merely moving a small aperture and photometer or the like lat- .erally so as to respond selectively to the scattered or diffracted light intensities at different. angles of deviation from the optical axis, as. has previously been suggested. The reason for such an advantage result-s from the fact that while the width of slit of the annular aperture 48 can be kept small so as to give a measurement at an exact angle of deviation, nevertheless a comparatively large amount of light at such angle is provided by the hollow cone of light available since 0, 0 and 0 are all substantially equal. On the other hand, if a small scanning aperture of a diameter equal to the width of the aperture 48 (in order to give an accurate indication) were used for scanning the difiracted light the intensity of the light being measured would be by comparison very small.

The present apparatus, on the other hand, due to the use of an annular light aperture instead of apin hole or the like, has the distinct advantage of admitting a far greater amount of light all rays of which have substantially the same common angle of scattering or deviation 0. Another important advantage which is also obtained from the optical arrangement employed is the fact that even though the scattered light intensity decreases rapidly as greater angles of scattering or deviation 0 are considered, nevertheless, the amount of light reaching the photomultiplier is appreciably compensated at such times due to the fact that the particles of the specimen scattering the light are being moved closer to the aperture 48.

In actual practice, it has been found desirable to construct the apparatus so that the aperture 48 will have a mean radius R of approximately 14 mm. and so that the near and far positions 22 and 24, respectively, will be axially displaced from the diaphragm 42 approximately 20 mm. and 200 mm. respectively. (Other values for R for such anarrangement when other materials are being tested mayrange between 5 and 20 millimeters.) Furthermore, while a slit width of .5 mm. has been used successfully with a mercury green radiation for the annular aperture 48, other slit widths up to 4.0 mm. may be satisfactorily employed depending upon the available intensity of the mercury, cadimum, hydrogen, sodium or other light source and color selected in accordance with the material being tested. However, for best resolution, the narrower slit widths are preferred. If a width of pin hole aperture of 0.3 mm. is used vw'th an objective having aaaaeos a magnification ratio of approximately 2 then an image of the pin hole 32 will be formed by the rays which will be approximately 0.6 mm. in width. The reason for selecting a mean radius of 14 mm. for the light aperture 48 is so that angular valves of light deviation between 5 degrees and 20 degrees will be provided the light rays being acceptable through the aperture 48; it being known that such angular values in a difiraction pattern for blood specimens, for example, are the values of most significance. Of course, a greater operating range can be employed if other degrees of deviation are to be recorded, and a range from approximately 1 /z to 45 is possible.

In Fig. 4 is shown a graph having along the abscissa thereof lineal values for variable axial distance h expressed in millimeters from 20 to 200 while along the ordinate thereof linear values for the amplified photo tube current I expressed in milliamperes from 0.0 to 1.4 are given. Upon this graph is shown a curve 96 like one actually traced by the apparatus of Fig. 1 while using monochromatic light at a wavelength of approximately 546 millimicrons and a sample of substantially normal blood. From this curve, it will be seen that a first dark ring occurs in the difiraction pattern when the sample is approximately 116 mm. from the diaphragm 42, and that a first bright ring occurs when the sample is approximately 77 mm. from the diaphragm. (It is also interesting to note that a second dark ring appears when the sampleis approximately 58 mm. from the diaphragm and a second bright ring appears when the sample is approximately 45 mm. therefrom.) Other blood samples having differing physical properties, will give curves having difiering characteristics on such a graph.

As previously stated, all of the light rays of the beam 39 in Fig. l which are undeviated by the small particles are directed so as to impinge upon the opaque disc 44. If the construction is considered geometrically, it will be seen that that part of the light from any single illuminated small particle in the specimen which is scattered so as to pass as a hollow cone of light through the annular aperture 48 will have an angle of deviation substantially equal to 0. At the same time the mean radius of the aperture will be R and the axial distance from the plane of aperture 48 to the specimen plane will be h. Thus, for all light which passes through the aperture 48 the geometric relationship which exists can be expressed as follows:

Red blood cells of a specimen are of various sizes and of irregular shapes; It is known, however, that they may be rendered spherical in shape Without change in volume by the use of a special diluting fluid containing a small amount lecithin (see Hemolysis and Related Phenomena, by E. Ponder, published in 1948 by Grune and Stratton, New York, N.Y.). Red cells in the spherizing medium at approximately one-half to one part of blood and diluted in 100 parts of a suspension fluid may be used and placed in a conventional microscope mold counting chamber which will thus provide a specimen having a depth of mm. and an area of approximately 20 mm. or more in diameter. Under such conditions the spherized cells will rest upon the bottom of the chamber and the random spacing thereof is such that a diffraction pattern will be produced which is independent of concentration.

The scattered light passing through the aperture 48 will be received by the phototube 56 and the current I for displacement of the recorder in the direction 76 will be proportional to the light intensity I scattered in the direction of the aperture 48 by the blood cells and inversely proportional to the square of the distance. Although the numbers of cells illuminated at different distances in the convergent beam are difierent, nevertheless, the change in number of cells illuminated at differsin 0 8 V ent distances is exactly compensated by the change in the light intensity thereon and thus the scattered light intensity in any direction will be constant; as if a constant number of cells are being illuminated by a constant amount of light. Thus The displacement of the table 66 and of the graph carried thereby will be linearly proportional to h. Thus, the motion of the element 70 is proportional to the intensities of the difiraction pattern multiplied by the factor (h +R Since the sin 2 0 is equal to this factor becomes proportional to sin 2 0.

If the particles in suspension are spheres of a radius r and of an index of refraction h and if the suspension fluid has an index of refraction n and if the ratio In of these two indices is close to unity, then the light intensity I scat-tered by the spheres in the direction can be expressed as follows:

I=nF(0, m, A, r) (3) wherein n is the number of spheres and )r is the wavelength of the light employed.

The amplitude A of such scattered light is given by a formula disclosed by Van de Hulst in an article Optics of Spherical Particles appearing in a Dutch publication Recherches Astronomiques de lObservatoire dUltrecht" by N. V. Drukkerij, Amsterdam, 1945 and may be written as follows:

f( 1+el con 0 -1 sin BEdEd -rr and wherein =='r sin 7 cos 5 (5) and =r sin 'y sin 45 (6) and wherein p and are variables of integration and vary from 0 to and from 0 to 360 respectively, and C is a constant depending on the Wavelength of the light used and on the number and radius of the spheres being considered.

The function F then will be given by the sum of the squares of the real and imaginary parts of the amplitude A. I

In the Expression 3 the variables m, r, A and 0 appear in the following combinations and wherein the wavelength is measured in the suspension fluid. Since it is more convenient to measure the wavelength in air, the Expression 3 may be rewritten as follows:

I=F(Z, p) and wherein 21r1' sin 6 Z- air (10) and for An=n,-n,,

21r21' (An) X air (11) It will be appreciated that particles such as blood in addition to scattering also absorb light in accordance with the wavelength used, so that the Expression 3 in order to give the right results for the cases where absorption is present will have to be modified so as to take into assume.

account. this effect If the. diffraction integral 4) is modified to include absorption, the amplitude value will be:

wherein a u and Act are extinction coefiicients of the surround, the sphere or particle-respectively and Once Equation 12 has been integrated, the intensity is equal to:

=G( p, )v

wherein Z and p are, given in EquationslO and 11 and has A In the case of :red blood cells,.K. may be assumed to be approximately constant for a particular wavelength; since n =Em r+ o where D and E depend on wavelength only and [Hb] is the concentration of hemoglobin in the blood cells. The index of the red cells has been found to, be very closely given by;E[b]+n since no does not difier substantially from the index of the suspension fluid mentioned supra, both n and n being close to the index of refraction of water.

Assuming :0, which is always the casewiththeliquids. here used the following can be written:

Thus, the preceding formula shows K to be dependent upon and substantially constant for any given wavelength.

It will be clear from the preceding description that Equation 13 solves the direct problem of finding the relative light intensity scattered by a. sphere of known characteristics and relative index of refraction close to unity and in a direction 0 relative to the incident light.

Due. to. the geometric arrangement employed, the light as seen by the phototube will be proportional to the inverse square of the distance between any particle in thespecimen: and any point in the. annular aperture. This inverse square distance is proportional to sin 0 and since for a given sphere the radius r is a constant, the inverse square distance Will be proportional to Z and the scattered light intensity as used will be given by the following:

Equation 16 will now permit us to predict the curve (see Fig. 4) which will be obtained by the instrument if An, r, K, A were given. However, to obtain useful information from the instrument, we must solve an. indirect problem; that is, given a curve as in Fig. 4 what are the characteristics r and An of the spheres under investigation which will give such a curve? This question can be answered with the following procedure. For a given color, the value of Kin, Equation is determined from the known. values. of the extinction coefiicients of; the, blood cells, the extinction coefficient of the surround a being equal to.zero.for all visible wavelengths.

For simplicity K is selected: equal to zero for the cadmium red line. )\=644 millimicrons (since for this color u =0). With this fixed value for K, a family of curves I versus Z can be plotted for any value of p by means of Equation 16. One such curve for p=3 and Z from 2.6 to 6 .4 is shown in Fig. 6. Other curvfisfor taken as equal to 0.64.4 i.

other values; of p could also have been plot r d on this, i e. i ired. Anumb r. o cu v s. e hatshawn in Fig. .6) have been plotted for discrete values of 4 between 3. and, 5.8. Allof these curvespresent one minimumand one maximum for certain values of 2 dep n ing upon the val of p employed.

Using; all of the. curves obtained for the different values of p, a plot ofp versus Z minimum (i.e. for all values of Z for which a minimum of 1Z occurs in Fig. 6,) and p versus Z maximum are obtained as shownat 122 and 124 in Fig. 7.

By inspection of Fig, 7, it will beseen that the curve 126 for the ratio Z to Z determines uniquely (within certain limits) the three values Z Z and p. Since Ihe rat o zmin min can be measured directly, the right hand quantities being given by the experimental curve, it is possible, by the use. of Fig. 7 and charting from the experimental ratio value, -to find Z and p.

Once Z hasbeen found r can be determined from Equation 10. Knowingythe values for r and p, the value for An can be determined by solving Equation 11. Thus the useful information r and An are. obtained.

In order to avoid the laborious procedure of obtaining the diarneter'and the An values for the spheres or cells from the curve of Fig. 4 by using the curves on Fig. 7, the curves of Figs. 8 and 9 have been plotted. In Fig. 8, there is shown a chart wherein a family of curves .128 for values, for h in millimeters (along the ordinate) and diameter values of particles in microns. (along the abscissa), the wavelength for such purpose having been The construction of Fig. 8 was determined in the, following manner. Select .a certain value for h (which corresponds to a sin 0 of x min and a certain h value, for example 78 mm. (which corresponds to a sin O value of sin- 0 sin 0 and using the, procedure explained above relative to Fig. 7', the value, of the diameter of the spherical particle may be determined from the curves 122 or 124 and 126 in Fig. 7. A point corresponding to h and diam eter of sphere is entered in Fig. 8; and this procedure is repeated until a sufiicient number of h points at suitable intervals have been established, Without changing the h value. All the points with the same h form a curve, such as curve 130.

Fig. 9- shows a plot of k versus diameter of spheres for different values of An. This figure was constructed by first selecting a value of An, for example .075. Once An is established, the h cor-responding to selected values of the diameters of the spheres can be calculated using curve 122 of Fig. 7, and the corresponding values of p obtained by using Equation 11 to find the value of Z Once 2 is determined, Equation 10 maybe solved for sin 0 and by llSing cot 6: 2

obtain h By keeping A31 constant and varying h a (16.5 mm.) was selected in such a way as to keep Z, one

eems 11 plurality of points may be determined. Thus a curve connecting a seriesof these points may be drawn and labeled with the appropriate value of An, and one such curve is designated by the numeral 132 in Fig. 9.

In the curve of Fig. 8, it is possible to enter immediately the value of h and the value of h and obtain therefrom the diameter of the spheres or cells. If a value for An is also desired, then in Fig. 9 enter the values for h and the diameter so determined from Fig. 8 to obtain a value for An. Such An values are of 10 clinical significance since they are believed to be closely related to the specific hemoglobin concentration of the blood cells (see Equation 14b). i

It is possible to combine the information of .Figs. 8 and 9 into a single graph in several different ways and 15 one such way is shown in Fig. 10. In this figure enter the cot 0 value for the first dark ring min) cot; 9 R

along the abscissa and proceed vertically until the correct value for cot a for the first bright ring mnx is reached in the first family of curves designated by the numeral 134 and from the point indicated thereby obtain a value for the volume of the spheres on the ordinate of the graph and obtain a An value from the other family of curves indicated by the numeral 136.

This graph has been scaled so that for an instrument value of R, the abscissa has the same scale as the traced curve. The curved lines for the maximums of the traced curves have been connected with the corresponding values ofthe abscissa so that cot fl scale also corresponds to the instrument tracing. In this arrangement, the computation of volume and refractive index may be carried out completely by graphical means without any computation or reference to numerical values. A convenient arrangement for this graphical procedure would be to overlay the properly scaled transparency of Fig. 10 on the traced curve. One could then follow lines from the minimum and maximum of experimental curve to an intersection point which would directly indicate values of volume and index difference, An.

While the curves of Figs. 7, 8 and "9 have been plotted for transparent spheres using R equal to 16.5 mm. and A equal to 644 m (for which radiation blood cells are transparent and therefore K may be taken as equal to zero), on the other hand the curves of Fig. 10 have been plotted for absorbing spheres since the mercury green line (546 mg) was used in this case and therefore K was taken as equal to 0.083.

It is desirable for visual comparison purposes to keep curves similar to the curve shown in Fig. 4 (when obtained from sphere of a similar kind but while using a radiation of a different wavelength for illumination) as independent as possible of wavelength. Accordingly, the radius R of the aperture 48 in the preceding paragraph of the parameters on which such curves depend, independent of wavelength. Since and wherein will not depend appreciably on A if is kept constant. practice R is made equal to 14 mm. X 644 546 Of course, 546 and 644 are the wavelengths in me of mercury green and cadmium red radiations respectively.

When the volume of individual blood cells can be determined as described above, this information can be used with hematocrit values of the blood specimens to give other useful information, such as the number of cells per unit volume of whole blood.

In order to obtain an estimate of size distribution of spherical particles or spherized cells, the mean size is first determined by the above described procedure. Then a curve of the specimen under consideration is compared with the theoretical curve for spheres of average size. The difference in modulation between these two curves will be an indication of the size distribution. (The word modulation as used herein is intended to mean the ratio of difference of the intensities at the first maximum and the first minimum to the sum of the same values.)

The instrument may also be used to obtain an indication of mean diameter and diameter distribution of cells which have not been spherized but which are contained in a known suspension fiuid; for example, red cells in suspension in their own serum. The procedure to obtain a curve from the instrument is the same as with spherized cells but the interpretation of this curve to obtain an indication as to mean diameter and diameter distribution is accomplished in such cases by comparing these curves with blood cell curves of known characteristics. Of course, a correct value for mean cell volume cannot be obtained from diameters thus obtained since the cells will not be of spherical form. The diameter distribution indication obtained in this way may be due both to actual variations in diameters, amounts of departure from spherical shape, or various combinations of these two factors.

Having described our invention, we claim:

1. Apparatus for use in determining the average size of a relatively large number of microscopic particles of varying sizes distributed at random in a specimen, said apparatus comprising a light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimenin said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, means for automatically indicating the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and means for indicating the position of said specimen with reference to said annular aperture for each intensity.

2. Apparatus for use in determining the average size of a relatively large number of microscopic particles of varying sizes distributed at randomin a specimen, said apparatus comprising a light source of relatively small size, an objective and opaque, means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, and means for automatically graphically producing a record indicative of the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and means for simultaneously graphically indicating on said record the position of said specimen with reference to said annular aperture for each intensity so recorded.

3. Apparatus for use in determining the average size of a relatively large number of microscopic particles of varying sizes distributed at random in a specimen, said apparatus comprising a light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, means for automatically graphically producing a record indicative of the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and means for simultaneously graphically indicating on said record a value indicative of the angle of deviation of the light rays for each intensity so recorded.

4. Apparatus for use in determining the average size of a relatively large number of miscroscopic particles of varying sizes distributed at random in a specimen, said apparatus comprising a light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, means for automatically producing upon a recording surface a specimen curve which represents in one predetermined direction thereon the various intensities of the light rays passing through said aperture for each position of said specimen and in a different predetermined direction thereon represents the various different operative positions of said specimen as said specimen is moved from a first extreme operative position to a second extreme operative position in said beam, and graphic means having a family of curves thereon for indicating in accordance with said intensities of the light rays passing through said aperture and in accordance with the position of said specimen for each intensity the mean size of the microscopic particle in said specimen, the curves on said graphic means and on said recording surface being of the same scaler value so that they may be disposed in adjacent relation to each other, at least one of said graphic means and said recording surface being transparent so that the intersection of said specimen curve with said family of curves when adjacent thereto can be readily observed and a scale value for mean size of particle corresponding thereto indicated on said graphic means.

5. Apparatus for use in determining the average size of a relatively large number of microscopic particles of varying sizes distributed at random in a specimen and the difierence in refractive indices between said particles and their surround, said apparatus comprising a light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size there in arranged in concentric relation with said opaque means and located substantially in conjugate relation to said light source, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said light beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, means for automatically producing upon a recording surface a specimen curve which represents in one predetermined direction thereon the various intensities of the light rays passing through said aperture for each position of said specimen and in a different predetermined direction thereon represents the various different operative positions of said specimen as said specimen is moved from a first extreme operative position to a second extreme operative position in said beam, and graphic means having a family of curves thereon for indicating in accordance with said intensities of the light passing through said aperture and in accordance with the position of said specimen for each intensity the mean size of the microscopic particle in said specimen and the difference in refractive indices between said particles and their surround, the curves on said graphic means and on said recording surface being of the same scaler value so that they may be disposed in adjacent relation to each other, at least one of said graphic means and said recording surface being transparent so that the intersection of said specimen curve with said family of curves when adjacent thereto can be readily observed and a scale value for mean size of particle and for refractive index diflerence corresponding thereto indicated on said graphic means.

6. Apparatus for use in determining the average size of a relatively large number of red blood cells of varying sizes distributed at random in a specimen, said apparatus comprising a monochromatic light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of relatively narrow width and predetermined size therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimen of blood in said beam, means for moving said holder and specimen toward and away from said annular aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by the red blood cells in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, and means for automatically graphically producing a record indicative of the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and means for simultaneously graphically indicating on said record the position of said specimen with reference to said aperture for one or more of the intensities so recorded.

7. Apparatus for use in analyzing a relatively large number of microscopic particles of varying sizes distributed at random in a specimen, said apparatus comprising a light source of size, an objective and a relatively small opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of small predetermined size and narrow width surrounding said opaque part, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said aperture While illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, the mean radius of said annular aperture and the displacement range of said holder and specimen being such as to cause scattered light rays of angles of deviation of values ranging between approximately 2 and 30 degrees to selectively pass through said aperture as said holder is moved through its operating range, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, means for automatically indicating the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and for simultaneously indicating the position of said specimen with reference to said aperture for each intensity.

8. Apparatus for use in determining the average size of a relatively large number of microscopic particles of varying sizes distributed at random in a specimen, said apparatus comprising a light source of relatively smallv size, an objective and opaque means arranged in spaced optically ahgned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of predetermined size surrounding said opaque part, a holder for positioning a specimen in said beam, means for moving said holder and specimen through a displacement range toward and away from said aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, the mean radiu of said annular aperture being in the neighborhood of 14 mm. and said aperture being of relatively narrow width so as to allow only a thin ring of light to pass therethrough at any time, the mean radius and width of said annular aperture and said displacement range of said holder and specimen being so related thereto as to cause scattered light rays of angles of deviation of values ranging between approximately 2 and 30 degrees to selectively pass through said aperture as said holder is moved through said displacement range, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays-so received, means for automatically indicating the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, and for simultaneously indicating the position of said specimen with reference to said aperture for each intensity.

9. Apparatus for use in comparing a scattered light intensity curve of microscopic particles of unknown characteristics with a curve from particles of known characteristics said apparatus comprising a light source of relatively small size, an objective and light intercepting means arranged in spaced optically aligned relation with each other, said objective being positioned so as to re ceive light from said light source and direct same as a convergent beam onto a small central portion of said light-intercepting means, said light-intercepting means having an annular aperture of predetermined size and narrow width in concentric relation with said central portion, a holder for positioning a specimen in said beam, means for moving said holder and specimen toward and away from said aperture while illuminated by said beam, whereby certain light rays of said beam will be variously scattered by particles in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, and means for automtically graphically producing a record indicative of the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, whereby said record may be visually compared with a curve of known characteristics.

10. Apparatus for use in determining the average size of a relatively large number of red blood cells of varying sizes distributed at random in a specimen, said apparatus comprising a monochromatic light source of relatively small size, an objective and opaque means arranged in spaced optically aligned relation with each other, said objective being positioned so as to receive light from said light source and direct same as a convergent beam onto an opaque part of said opaque means, said opaque means having an annular aperture of predetermined size and narrow width therein arranged in concentric relation with said opaque part and located substantially in conjugate relation to said light source, a holder for positioning a specimen of blood in said beam, means for moving said holder and specimen toward and away from said diaphragm while illuminated by said beam whereby certain light rays of said beam will be variously scattered by the red blood cells in said specimen, electrical means for receiving that part of the scattered light rays passing through said aperture and for producing a signal the instantaneous value of which is proportional to the intensity of the light rays so received, and means for automatically graphically producing a, record indicative of the various intensities of the light rays passing through said aperture for each position of said specimen as said specimen is moved from a first operative position to a second operative position in said beam, means for simultaneously graphically indicating on said record the position of said specimen with reference to said aperture for each of the intensities so recorded, and transparent means having a graph thereon arranged to overlie said record and sealed in accordance with the size of the aperture employed in said apparatus and the range of movement of said specimen holder for providing mean cell size values and refractive index difierence values of the specimen in accordance with the curve traced on said record. I

11. The combination as defined in claim 10 and wherein the size and width of said annular aperture are so '18 related to the range of movement of said holder as to successively admit through said aperture scattered light at all angles between approximately 5 degrees and 20 degrees for collection by said electrical means.

References Cited in the file of this patent UNITED STATES PATENTS 2,065,114 Cahn et a1. Dec. 22, 1936 2,519,997 Brown Aug. 22, 1950 2,769,365 Loeschcke et al Nov. 6, 1956 2,788,702 Baum Apr. 16, 1957 

